Acta Bot. Croat.

79 (1), 15–25, 2020 CODEN: ABCRA 25

DOI: 10.37427/botcro-2020-002 ISSN 0365-0588
eISSN 1847-8476

Preliminary characterization of the Quercus

pubescens complex in southern Italy using molecular
markers
Romeo Di Pietro1, Piera Di Marzio2, Gaby Antonecchia3, Antonio Luca Conte2, Paola Fortini2*
1
 epartment of Planning, Design, Architecture technology (PDTA), Herbarium Flaminio, Sapienza University of Rome,
D
Rome, Italy
2
Department of Bioscience and Territory, University of Molise, Pesche (IS), Italy
3
Ministry of Education, University and Research (MIUR), Rome, Italy

Abstract – Quercus pubescens s.l. is a group of taxonomically intricate and highly debated deciduous white oaks
widely distributed in southern Europe. The Apulia region occupies the south-easternmost part of the Italian Pen-
insula; the land-use pattern is based on extensive agricultural systems and only 10% is covered by forests that are
mainly composed of oak woods. It is the region in Italy showing the highest number of oak species, among which
four putative species of the Quercus pubescens group, have been reported in floras and checklists with uncertain
taxonomic value because of the overlapping of diagnostic characters. In this paper, we carried out a molecular
analysis on natural populations of Q. pubescens s.l. distributed throughout the Apulia region. Individuals from 24
pubescent oak populations were sampled and each tree was genotyped at 11 polymorphic microsatellite markers.
Overall, the average expected heterozygosity (He) was 0.629, and the allelic richness (Ar) ranged between 2.130
and 7.187. No differentiation was observed among the populations investigated, and the genetic differentiation
coefficient (FST) was 0.036. Gene flow among populations was found to be relatively high (Nm = 6.664). From a
taxonomic point of view, the possibility of the coexistence of more than one species among the Apulian pubes-
cent oaks reported in the taxonomic and syntaxonomic literature is not supported by the results of this molecular
analysis.

Key words: genetic diversity; Italy; population structure; Quercus pubescens; SSR; taxonomy

Introduction
The genus Quercus L. subgenus Quercus has a wide dis-
tribution in the Northern Hemisphere, especially in central
and southern Europe, where Quercus species form important
forest communities in the Temperate and the Mediterranean
bioclimatic regions (Nixon 1993, Govaerts and Frodin 1998).
Italy is the country showing the highest oak species di-
versity in Europe, although there is still incomplete agree-
ment on the exact number of oak taxa occurring in the ter-
ritory. Most of the taxonomic uncertainty regarding the
Italian (and European) oaks concerns the so-called white
oaks and in particular the pubescent oaks (Quercus pube-
scens s.l. subgen. Quercus Sect. Quercus). The most recent
Italian flora and checklists (Pignatti et al. 2017, Bartolucci et
al. 2018) report various pubescent oaks in southern Italy (Q.
amplifolia Guss., Q. apennina Lam., Q. congesta C. Presl, Q.
dalechampii Ten., Q. leptobalana Guss., Q. ichnusae Mossa,
Bacch. et Brullo, Q. humilis Mill., Q. virgiliana Ten.) which
are all considered Steno-Mediterranean vicariant species of
Q. pubescens Willd.
Apulia is the easternmost Italian administrative region
and all Italian oak species, both evergreen and deciduous,
occur in this region with the exception of Q. petraea (Matt.)
Liebl. Despite this richness in oak species and a wide poten-
tial range for the oak woodlands, only 10% of the Apulian
territory is covered by forests. The Q. pubescens s.l. forests
are the most widespread and can be found in the flooded de-
pressions of the plain, in the limestone plateaus and on the
sub-montane rocky slopes.
Molecular marker studies have not yet been published
for the genus Quercus in the Apulian region. However, the

* Corresponding author e-mail: fortini@unimol.it

ACTA BOT. CROAT. 79 (1), 2020 15

DI PIETRO R, DI MARZIO P, ANTONECCHIA G, CONTE AL, FORTINI P
evaluation of genetic variation can be of great importance
for the conservation and management of forest ecosystems,
especially in areas particularly vulnerable to climatic change
(Peñuelas et al. 2017) and in forests whose original extent has
been significantly reduced. This is the case of the oak forests
of the Apulia region. In fact, the dry Mediterranean climate
that characterizes this region places it at risk of desertifica-
tion if the climate should experience a further increase in
aridity. Moreover, the widespread practice of extensive ag-
riculture (wheat, olive and vine) during the last century led
to the destruction of most of the natural forest resources
(Biondi et al. 2010).
Several reports on the population genetics of the white
oaks Q. robur L., Q. petraea, Q. pubescens have been pub-
lished for south-eastern Europe in the last two decades
(Franjić et al. 2006, Curtu et al. 2007a, Jerše and Batič 2007,
Slade et al. 2008, Curtu et al. 2009, Ballian et al. 2010, Enes-
cu et al. 2013, Gailing et al. 2013). In Italy, only few data are
available. Fineschi et al. (2002) and Fineschi and Vendramin
(2004) presented a study on the genetic diversity of the Ital-
ian white oaks analyzing chloroplast DNA. Subsequently,
several studies that compared leaf morphology and molecu-
lar data on some white oaks populations of Central Italy have
been published (Fortini et al. 2009, 2013, 2015).
In the most recent papers published on the Apulia forests
(Biondi et al. 2004, Di Pietro and Misano 2009) the follow-
ing pubescent oaks were reported in the phytosociological
tables: Q. pubescens, Q. virgiliana, Q. amplifolia and Q. dale-
champii. A recent study (Di Pietro et al. 2016) statistically
analyzed 25 morphological characters of leaves and fruits
within 24 pubescent oak populations in the Apulia region,
in order to identify possible diagnostic traits useful for taxo-
nomic differentiation. The results provided no evidence for
differentiation among tree individuals based on compari-
sons of morphological traits. In the present study plant ma-
terial from the same pubescent oak populations was used to
analyze their genetic diversity, structure and gene flow. The
aims of this study are twofold. First, to assess whether any
group of genetic structure could be identified among oak
individuals and/or populations, notwithstanding the mor-
phological and taxonomical uniformity highlighted in the
previous morphological work (Di Pietro et al. 2016). Second,
to investigate the pattern of genetic diversity in the Apulian
pubescent oaks in order to provide useful information for
further studies.
Material and methods
Study area
The Apulia region is located in the south-eastern part of
the Italian Peninsula where it is largely open to the Adriatic
and Ionian Seas. It is slightly sloping, with more than 60%
of the territory occurring below 200 m a.s.l. Five physio-
graphic units can be distinguished in the Apulia region and
these are the Daunian sub-Apennine (1150 m), the Garga-
no promontory (1080 m), the Murgian Plateau (680 m), the
Salento Peninsula and the large plain Tavoliere delle Puglie.
16 ACTA BOT. CROAT. 79 (1), 2020
The bedrock of the mountainous systems is mainly com-
posed of Cretaceous limestone and Paleocenic calcarenites.
Marls occur in the lower parts of the Daunian sub-Apennine
and in the Tavoliere plain together with conglomerates and
Pleistocenic and Holocenic sand deposits. The bioclimate is
Thermo-Mediterranean and Meso-Mediterranean with the
Temperate region occurring only in the sub-and lower-mon-
tane belts. The rainiest areas are the Gargano, the Daunian
sub-Apennine and the south-eastern part of the Salento Pen-
insula (slightly over 800 mm year–1). Annual precipitation
values on average of less than 500 mm year–1 are recorded
in the western side to the Murgian Plateau and in the Tavo-
liere plateau whereas the remaining portion of the territory
exhibits an average annual rainfall of between 500 and 700
mm year–1 (Blasi and Michetti 2007, Cotecchia et al. 2014).
As far as the climax vegetation is concerned almost the en-
tire Apulian territory would potentially be covered by oak
forests except for some scattered coastal and subcoastal ar-
eas dynamically linked to the Pinus halepensis Mill. woods
and maquis, and the inner part of the Gargano promontory
where the wide “Foresta umbra” beech wood occurs (Biondi
et al. 2004, 2010).
Population sample information
Individual mature oak trees were randomly selected in
each population trying to maintain a distance of at least 20
meters from each other. Leaves were randomly collected
from 379 individuals within 24 populations (subdivided
in squares 50 × 50 m), which covered approximately uni-
formly the physiographic units of the Apulia region (Fig.
1, Tab. 1, On-line Suppl. Tab. 1). Both the individuals and
the populations they belong to are the same on which the
morphological analysis of leaves and acorns was performed
in the paper by Di Pietro et al. (2016). The collected oak
specimens were dried on silica gel, and stored at room tem-
perature until analysis. Voucher specimens for each indi-
vidual sampled were stored at the Herbarium of the Uni-
versity of Molise.
DNA extraction, EST-SSR amplification
Total genomic DNA was extracted from 0.5 g of dried
leaves from all samples using spin columns of “Invisorb®
Spin Plant Mini Kit” and following the protocol of the man-
ufacturer.
Eleven microsatellite loci or simple sequence repeats
(EST-SSRs), developed by Durand et al. (2010) (PIE020,
PIE102, PIE152, PIE215, PIE223, PIE227, PIE239, PIE242,
PIE243, PIE267, PIE271) and polymorphic in white oaks
(Guichoux et al. 2011, Antonecchia et al. 2015) were se-
lected. Polymerase chain reaction (PCR) amplification was
performed in a single multiplex reaction in a DNA Engine
Tetrad (MJ Research Bio-Rad) thermocycler and PCR prod-
ucts were run on an ABI 3730xl capillary sequencer (Applied
Biosystems) using Genescan 600 LIZ internal size standard
(Applied Biosystems). Alleles were scored using STR and
software version 2.3.106 (Toonen and Hughes 2001) and al-
 QUERCUS PUBESCENS COMPLEX IN SOUTHERN ITALY

Fig. 1. Locations of the 24 oak collection stands.

Tab. 1. Quercus species composition of the 24 oak populations (according to Biondi et al. 2004, Biondi and Guerra 2008, Di Pietro and
Misano 2009) with number of genotyped individuals and coordinates.
Population Analyzed Genotyped
Latitude (N) Longitude (E) Quercus species
code individuals individuals
Pop01 16 16 4524805 650840 Q. pubescens
Pop02 16 16 4557220 526723 Q. pubescens
Pop03 16 16 4624669 567854 Q. pubescens
Pop04 16 16 4624043 561581 Q. virgiliana, Q. dalechampii, Q. pubescens
Pop05 16 15 4562416 534533 Q. virgiliana, Q. dalechampii, Q. pubescens
Pop06 16 15 4564231 529921 Q. pubescens
Pop07 16 16 4558496 530175 Q. pubescens
Pop08 16 16 4512469 618357 Q. pubescens
Pop09 18 16 4497035 647716 Q. pubescens
Pop10 16 16 4582747 553553 Q. pubescens, Q. dalechampii, Q. virgiliana
Pop11 16 16 4551101 622237 Q. virgiliana, Q. dalechampii, Q. amplifolia
Pop12 15 15 4504016 656599 Q. pubescens
Pop13 12 12 4491270 658881 Q. pubescens, Q. dalechampii, Q. virgiliana
Pop14 16 16 4446957 278106 Q. virgiliana, Q. amplifolia, Q. dalechampii
Pop15 15 15 4514201 647317 Q. pubescens
Pop16 16 11 4502657 686693 Q. pubescens
Pop17 16 13 4534971 632507 Q. pubescens, Q. dalechampii, Q. virgiliana
Pop18 16 14 4523011 649110 Q. dalechampii, Q. virgiliana, Q. pubescens
Pop19 16 14 4499728 654902 Q. pubescens
Pop20 17 14 4635503 582284 Q. pubescens, Q. dalechampii, Q. virgiliana
Pop21 16 16 4504890 744270 Q. virgiliana, Q. dalechampii, Q. pubescens
Pop22 14 11 4518821 650238 Q. virgiliana, Q. dalechampii, Q. pubescens
Pop23 16 6 4540867 597285 Q. pubescens
Pop24 16 7 4635677 587298 Q. pubescens

lele binning was performed as described in Guichoux et al.
(2011). MICROCHECKER (Van Oosterhout et al. 2004) was
used to check for the presence of null alleles and to check for
potential problems related to allele dropout with 1000 ran-
domizations and a 95% confidence interval.
Data analysis
Polymorphic EST-SSR markers were used to analyze the
genetic diversity of 338 Q. pubescens s.l. individuals in 24
ACTA BOT. CROAT. 79 (1), 2020 17
populations. EST-SSR amplification was unsuccessful for 41
of the 379 individuals sampled. Two populations (Pop23 and
Pop24) were not included in the statistical analyses because
of the low number of amplified individuals.
The basic statistics for each locus: number of alleles
(NA), number of individuals (N), observed heterozygosity
(Ho), expected heterozygosity (He), and number of private
alleles (PA) were computed using the statistical program Ge-
nAlEx version 6.503 (Peakall and Smouse 2012).
DI PIETRO R, DI MARZIO P, ANTONECCHIA G, CONTE AL, FORTINI P

Polymorphic information content (PIC), deviation from
Hardy-Weinberg equilibrium (HW), and null allele frequen-
cy (F(Null)) were computed for each locus using Cervus
software version 3.0.7 (Kalinowski et al. 2007).
The basic statistics for each population: number of dif-
ferent alleles, number of effective alleles, allelic richness, ob-
served heterozygosity, expected heterozygosity, index of fix-
ation (FIS) calculated as F = 1 – (Ho / He) and number of
private alleles, were computed using Arlequin software ver-
sion 3.5.2.2 (Excoffier and Lischer 2010). Allelic richness per
locus and population were computed using FSTAT version
2.9.4 (Goudet 2003).
Analysis of molecular variance (AMOVA) was per-
formed using GenAlEx in order to partition the total mi-
crosatellite diversity, among and within populations, and
within the individuals of each Q. pubescens s.l. population,
based on 11 EST-SSR markers loci. The variance compo-
nents were statistically tested by non-parametric random-
ization tests of significance based on 1000 bootstraps. Gene
flow among populations was estimated using the indirect
method based on the number of migrants per generation
(Nm) using the formula, Nm = 0.25 (1 − FST) / FST (Slatkin
and Barton 1989). To investigate population differentiations,
pairwise FST between all pairs of populations was computed,
FST was calculated according to Weir and Cockerham (1984)
in program FSTAT 2.9.4.
Principal coordinate analysis (PCoA) with data stan-
dardization based on Nei’s genetic distance was performed
using GenAlEx.
Bayesian clustering analysis, using STRUCTURE soft-
ware version 2.3.4 (Pritchard et al. 2000, 2010), with popu-
lation IDs as Sampling Location Information, has been car-
ried out. We opted for the admixture with correlated allele
frequencies model between populations. We used a burn-in
period of 25,000 and a number of MCMC of 100,000 cy-
cles. Ten runs for each K (K = number of possible clusters)
value were done to test the consistency of the results. Each
(K) value was tested from K = 1 to K = 10. The optimum K
value was predicted using the web-based software STRUC-
TURE HARVESTER web version 0.6.94 which implements
the Evanno method (Earl and vonHoldt 2012). Individu-
als with probabilities above 0.80 (Q ≥ 0.800) were assigned
as putative purebred while all the other individuals were
assigned as “putative hybrids” although the term “hybrid”
could be a little misleading since no plant material of pos-
sible parental species was collected. For this reason we have
opted for using the much more neutral term “off-type” in
the rest of the text.
BOTTLENECK software version 1.2 (Cornuet and Lui-
kart 1996) was used to detect the likelihood of a bottleneck
effect. Heterozygosity excesses were displayed, identified
based on the estimates of multilocus genotypes calculated
by using the Wilcoxon signed rank tests and evaluating de-
partures from the ratio 1:1 deficiency/excess (Cornuet and
Luikart 1996, Luikart and Cornuet 1998, Piry et al. 1999).
A two-phase mutation model (TPM) and a stepwise muta-
tion model (SMM) for Wilcoxon signed-rank tests were used
considering a 90% SMM proportion and TPM with a vari-
ance of 10, and 1000 repeats.
Results
SSR polymorphism and genetic diversity
All the loci were polymorphic (Pj = q ≤ 0.99). The total
number of alleles identified was 100 out for the 325 individu-
als analyzed. The allele fragment sizes (Tab. 2) matched with
the relative SSR reference size, as described in Guichoux et
al. (2011). The number of alleles per locus (k) ranged be-
tween 4 and 17, with an average of 11 alleles. The PIC values
varied from 0.233 (PIE227) to 0.879 (PIE152). According to
(Hildebrand et al. 1992) eight EST-SSR loci were found to
be informative (PIC ≥ 0.5), whereas two were found to be
slightly informative (PIC < 0.4). The observed heterozygos-

Tab. 2. Allelic diversity of eleven microsatellite loci scored in 325 individuals of Quercus pubescens s.l.; S – size range fragments (bp);
NA – Number of alleles at the locus, Ar – Allelic richness per locus, N – Number of individuals typed at the locus, Ho – Observed
heterozygosity, He – Expected heterozygosity, PIC – polymorphic information content, HW – Significance of deviation from Hardy-
Weinberg equilibrium (Hardy-Weinberg equilibrium test chi-square value, p value and significance with Bonferroni correction: ns =
not significant, * = P < 0.05; ** = P < 0.01; *** = P < 0.001), F(Null) – Null allele frequency estimate, FIS – Fixation index (* = P < 0.05).
Locus S NA Ar N Ho He PIC HW F(Null) FIS
PIE020 97–109 7 3.386 312 0.583 0.585 0.537 35.434*** –0.026 –0.067
PIE102 139–167 13 5.367 321 0.523 0.750 0.726 87.243 *** 0.187 0.279*
PIE152 228–260 17 7.187 314 0.834 0.890 0.879 3.756 ns 0.032 0.043*
PIE215 185–218 12 6.295 281 0.384 0.707 0.673 193.668*** 0.311 0.417*
PIE223 197–234 11 5.980 318 0.811 0.822 0.803 0.525 ns 0.006 –0.002
PIE227 156–165 4 3.387 323 0.223 0.251 0.233 3.776 ns 0.060 0.095*
PIE239 69–93 9 4.689 302 0.248 0.435 0.420 56.089*** 0.274 0.399*
PIE242 102–132 14 5.924 318 0.748 0.841 0.821 16.677ns 0.058 0.093*
PIE243 204–224 10 4.392 282 0.688 0.683 0.649 37.002*** –0.029 –0.038
PIE267 84–106 12 4.029 313 0.425 0.667 0.622 82.591*** 0.227 0.334*
PIE271 182–204 12 2.130 311 0.949 0.843 0.823 37.040*** –0.063 –0.153
Mean – 11 – 308.64 0.583 0.679 0.653 – 0.094 –
Standard error – 1.053 – 4.386 0.074 0.058 0.059 – 0.040 –

18 ACTA BOT. CROAT. 79 (1), 2020

 QUERCUS PUBESCENS COMPLEX IN SOUTHERN ITALY

ity (Ho) for microsatellite loci ranged from 0.223 (PIE227)
to 0.949 (PIE271), and the average was 0.583. A high level of
Ho was detected in seven EST-SSR loci ranging from 0.523
to 0.949, whereas four loci detected low Ho with values rang-
ing from 0.223 to 0.425. The expected heterozygosity (He)
ranged from 0.251 to 0.890 with an average value of 0.679
and was higher than Ho for all the microsatellite loci, ex-
cept for PIE243 and PIE271. The Hardy-Weinberg exact test
for all populations (HW) revealed that seven loci (PIE020,
PIE102, PIE 215, PIE239, PIE243, PIE267, PIE271) exhib-
ited significant deviation from Hardy–Weinberg equilibri-
um (P<0.001) whereas four loci (PIE152, PIE223, PIE227,
PIE242) did not show significant departures from Hardy-
Weinberg equilibrium.
The indexes of genetic diversity of the 22 Q. pubescens s.l.
populations are summarized in Tab. 3. The number of differ-
ent observed alleles (Na) and effective alleles (Ne) averaged
across all loci ranged from 4.727 (Pop22) to 7.182 (Pop09)
and 2.559 (Pop22) to 4.363 (Pop20), respectively. Nine pri-
vate alleles (PA) was found and these are distributed within
ten individuals in turn distributed in eight oak populations
(two other private alleles were found in Pop24; On-line Sup-
pl. Tab. 2). The allelic richness (Ar), ranged between 3.865
(Pop3) and 5.171 (Pop20). The average observed heterozy-
gosity (aHo) ranged from 0.487 (Pop03) to 0.665 (Pop11) with
mean value of 0.583. The average expected heterozygosity
(aHe) ranged from 0.510 (Pop03) to 0.709 (Pop09) with mean
value of 0.629. Overall, the observed heterozygosity showed
mostly slightly lower values than the expected heterozygosity.
The mean fixation index (FIS) values are close to zero for all
populations (ranging from 0.058 in Pop11 to 0.188 in Pop21)
that are the values expected under a random mating.

Population genetic structure and gene flow

Tab. 3. Genetic diversity parameters for the Quercus pubescens The overall population differentiation degree is reported
s.l. populations (Pop) analyzed through eleven microsatellite loci. in Tab. 4. AMOVA analysis showed that 3.6% (P < 0.001) of
n – number of individuals; Na – average number of different al-
leles; Ne – average number of effective alleles; Ar – allelic richness; the genetic variations was among populations while 96.4%
aHe – average expected heterozygosity; aHo – average observed was within populations. The majority of molecular variance
heterozygosity; FIS – average of fixation index tested by 1023 per- was partitioned within individuals (78.5%, P < 0.01) while
mutation of gene copies between individuals within population
17.9% (P < 0.01) of variance was found to occur among in-
(* = P < 0.05); PA – number of private alleles.
dividuals. The pairwise FST matrix between populations is
Pop n Na Ne Ar aHo aHe FIS PA shown in On-line Suppl. Tab. 3; eleven population pairs ex-
Pop01 16 6.182 3.641 4.642 0.581 0.604 0.035 0 hibited negative values but all of them showed no significant
Pop02 16 5.091 3.353 4.064 0.498 0.584 0.125* 0 p values (all largely > 0.05). The differentiation coefficient
Pop03 16 4.818 2.968 3.865 0.487 0.510 0.029 0 between population pairs having significant P values showed
Pop04 16 6.364 3.684 4.605 0.572 0.638 0.094* 0
that Pop11 (Incoronata-Tavoliere) and Pop15 (Palmariggi-
Pop05 15 5.182 3.097 4.192 0.604 0.610 -0.036 1
Salento) was the smallest (FST = 0.0025; P ≤ 0.001); the dif-
Pop06 15 5.182 3.055 3.940 0.529 0.590 0.037 0
Pop07 16 5.636 3.213 4.156 0.557 0.544 -0.059 0 ferentiation coefficient between Pop02 (Deliceto-Daunian
Pop08 16 6.455 3.888 4.775 0.577 0.632 0.086* 0 sub-Apennine) and Pop22 (Vico del Gargano-East Gargano)
Pop09 16 7.182 4.166 4.903 0.605 0.709 0.155* 0 was the largest (FST = 0.1053; P < 0.01).
Pop10 16 6.455 3.979 4.601 0.587 0.656 0.093* 0 Nei’s genetic distance among the populations belonging
Pop11 16 6.000 3.369 4.313 0.665 0.614 -0.112 0 to the five units are graphically illustrated in Fig. 2, con-
Pop12 15 5.909 3.790 4.420 0.628 0.655 0.025 1 structed on the basis of principal coordinate analysis (PCoA
Pop13 12 5.545 3.883 4.760 0.595 0.705 0.092 2 with the physiographic units superimposed). The first two
Pop14 16 6.091 3.749 4.487 0.655 0.648 -0.018 1
axes of the PCoA explained 42.26% of the variation. The
Pop15 15 5.727 3.503 4.583 0.596 0.634 0.066 0
Pop16 11 5.273 3.400 4.606 0.627 0.647 0.006 1
scattergram did not show an evident correlation between
Pop17 13 6.091 3.700 4.560 0.580 0.639 0.062 0 the physiographic units and the genetic similarities of the
Pop18 14 5.909 3.688 4.651 0.605 0.642 0.005 1 populations. In fact, populations belonging to four differ-
Pop19 14 6.545 3.955 4.841 0.583 0.669 0.064 0 ent physiographic units are closely associated along the first
Pop20 14 7.091 4.363 5.171 0.617 0.692 0.120* 1 axis in the left part of the diagram. The Mantel test compar-
Pop21 16 6.000 3.753 4.510 0.557 0.684 0.116* 1 ing the matrices of geographic and genetic distances among
Pop22 11 4.727 2.559 3.941 0.527 0.540 -0.129 0 populations did not evidence any correlation between these
Mean 14.8 5.884 3.580 4.481 0.583 0.629 0.039 two variables.

Tab. 4. Analysis of molecular variance among populations, within populations, and within individuals of Quercus pubescens s.l. popula-
tions; Df – degree of freedom, SS – sum of squares, MS – mean squares, P – probability based on standard permutation across the full
data set, Nm –average estimate of gene flow among populations.
Source of variation Df SS MS Estimated variation Percentage variation % F-statistics P-value Gene flow
Among Populations 21 180.496 8.595 0.141 3.6 Fst = 0.036 Nm = 6.664
Among Individuals 303 1346.241 4.443 0.695 17.9 Fis = 0.185 0.001
Within Individuals 325 992.500 3.054 3.054 78.5 Fit = 0.215
Total 649 2519.237 3.889 100

ACTA BOT. CROAT. 79 (1), 2020 19

DI PIETRO R, DI MARZIO P, ANTONECCHIA G, CONTE AL, FORTINI P

netic clusters: a first cluster including 305 pure individu-

Fig. 2. Principal Coordinate Analysis (PCoA) via Distance ma-
trix (Nei Genetic Distance) with data standardization. Daunian
sub-Apennine populations: Pop02, 06, 07, 08; W-Gargano popu-
lations: 04, 05; E-Gargano populations: 21, 22; Murgia plateau
populations: 01, 03, 09, 10, 12, 13, 14, 16, 17, 18, 19, 20; Salento
population: 15; Tavoliere population: 11.
When STRUCTURE was run using population IDs as
sampling location information, a maximum value of the rate
of change in the log probability of data was revealed at K
= 2, using Evanno's method (Evanno et al. 2005) (On-line
Suppl. Fig. 1).
The individual membership proportion determined
by Bayesian clustering analysis suggested a subdivision of
the whole data-set of oak individuals (325) into two ge-
als (94.15%) and a second one composed of 19 off-type
individuals (5.85%) (Fig. 3a). The distribution of the 19
off-types involved 13 populations out of the 22 investigat-
ed (Fig. 3b, On-line Suppl. Tab. 4). Population 13, where
the sampled individuals were selected from a mixed wood
with Q. pubescens and Q. robur that developed in a humid
area, is the population showing the highest number (4) of
off-types. Populations 2, 9, and 10 count two off-types each
and relate to Q. pubescens s.l. woods with the occurrence
of Q. frainetto.
The Wilcoxon sign-rank test showed no significant re-
sults as regards bottleneck effect in all the populations an-
alyzed, using both TPM and SMM models. The observed
number of loci with heterozygosity excess (obs LHexc) was
found to be always lower than the expected number of loci
with heterozygosity excess (exp LHexc) except for popula-
tion 13 where “obs LHexc” is about 1.5 times greater than
“exp LHexc”. However, the Wilcoxon sign-rank test was not
significant (Tab. 5).
Discussion
The data-set exhibited the following levels of genetic di-
versity at the microsatellite loci examined: mean expected
heterozygosity (He) = 0.679, mean observed heterozygos-
ity (Ho) = 0.583, average number of alleles per locus (K) =
11.0 (from 4 to 17 alleles). Four (PIE102, PIE215, PIE239,
PIE267), out of the eleven loci investigated, showed a posi-
tive FIS (0.279-0417) which turned out to be statistically sig-
nificant (Tab. 2). It is known that the fixation of alleles could
be favored by directional selection (Andolfatto 2001), or be
the results of genetic drift. Molecular studies regarding oth-

Fig. 3. Genetic assignment obtained with STRUCTURE clustering analysis. Oak individuals are distributed in progressive order along
the horizontal axis while the vertical axis expresses the group membership percentage degree per single individual per K=2. Clustering
of all the data-set oak individuals into two different genetic clusters with decreasing membership degrees for group 2 and increasing
degrees for group 1 (a). Clustering of all the data-set oak individuals ordered per populations; the latter separated by white lines (b).

20 ACTA BOT. CROAT. 79 (1), 2020

 QUERCUS PUBESCENS COMPLEX IN SOUTHERN ITALY

Tab. 5. Results of tests for genetic bottlenecks using the two-phase the analysis of populations composed of a seemingly limited
mutation model (TPM) and a stepwise mutation model (SMM), number of samples, can still provide interesting information
and the Wilcoxon sign-rank tests for heterozygosity excess; that should not be completely ignored.
the number of polymorphic loci for all populations (Pop) is 11;
obs LHexc – observed number of loci with heterozygosity excess; The highest allelic variability was found in locus PIE152
exp LHexc – expected number of loci with heterozygosity excess; with 17 alleles and PIC = 0.879. A similar variability was
P – probability of no significant heterozygosity excess. found in other pubescent oak populations investigated in
TPM SMM southern Italy and central Italy (Antonecchia 2015) where
obs exp obs exp locus PIE152 exhibited the highest polymorphic degree and
Pop P P
LHexc LHexc LHexc LHexc in particular the highest values of allelic richness for Q. pu-
Pop01 4 6.46 0.9492 2 6.31 0.9954 bescens. The still insufficient knowledge about the Q. pube-
Pop02 6 6.44 0.7114 4 6.43 0.8398 scens genome do not allow it to be established if the high
Pop03 4 6.43 0.9385 4 6.43 0.9585
variability in locus PIE152 might be associated with adap-
Pop04 4 6.37 0.9126 4 6.37 0.9585
Pop05 3 6.29 0.8608 3 6.29 0.9263 tive traits. More in-depth investigations would be needed to
Pop06 5 6.39 0.7676 4 6.35 0.8799 clarify this point.
Pop07 4 6.36 0.9585 4 6.38 0.9731 The level of genetic variability within the pubescent oak
Pop08 5 6.49 0.9492 4 6.58 0.9663
Pop09 3 6.55 0.9492 2 6.50 0.9976
populations of the Apulia region was here found to be low-
Pop10 5 6.27 0.7676 3 6.36 0.8970 er than that reported for Q. pubescens in other countries.
Pop11 2 6.39 0.9895 2 6.49 0.9966 Curtu et al. (2007b) reported aHe = 0.891, Ar = 17.8 on a
Pop12 5 6.49 0.5845 5 6.57 0.7114 study based on 6 microsatellite loci for 73 Q. pubescens in-
Pop13 8 6.52 0.1602 8 6.61 0.2598 dividuals while Enescu et al. (2013) reported aHe=0.847, k
Pop14 4 6.59 0.8608 4 6.58 0.9585
Pop15 6 6.58 0.7676 5 6.51 0.8608 = 22 and Ar = 14.22 in a 7 microsatellite loci study in a geo-
Pop16 4 6.59 0.9263 4 6.55 0.9663 graphically related area. The heterozygosity values reported
Pop17 5 6.46 0.8799 2 6.51 0.9919 in these studies, however, would not be directly comparable
Pop18 3 6.47 0.9126 3 6.50 0.9126 with those obtained by us due to the use of different type of
Pop19 4 6.39 0.9126 4 6.50 0.9492 markers (genomic SSRs vs. EST-SSRs). Some authors (Du-
Pop20 4 6.48 0.8799 3 6.59 0.9919
Pop21 6 6.56 0.6812 4 6.57 0.8970 rand et al. 2010, Parthiban et al. 2018) showed that the use
Pop22 3 6.56 0.9663 2 6.53 0.9954 of EST-SSRs highlights the values of polymorphism slightly
lower than those obtained from the use of genomic SSRs.
However, there are some studies on the Quercus genus in
which both these types of markers were tested. Curtu et al.
er oak species stated that the genetic variation removed by
(2011) investigated the genetic variability of 65 individuals of
genetic drift would affect the genome rather uniformly (Al-
Q. pubescens using 7 microsatellite loci, of which 5 gSSR and
berto et al. 2010). This would not seem to be the case of the
2 EST-SSRs. The overall average values for the genetic pa-
Apulian pubescent oak populations, since the other EST-SSR
rameters in issue were the following: aHe = 0.859, k = 17, Ar
loci examined showed only slightly positive values of FIS, or
= 16.5. Calculating the values of these parameters solely for
even negative ones. Due to the relatively low number of sam-
the two EST-SSRs markers we obtained values (aHe = 0.819,
pled individuals per population, it cannot be completely ex-
k = 17, Ar = 16.8) that are lower than those obtained aver-
cluded that these results could be addressed, at least in part,
aging both kind of markers but still significantly higher than
to the sampling effect. However, there is no uniformity of
those we obtained from our Apulian pubescent oak data-set.
views at present on the minimum number of individuals per
population that should be sampled to detect “reliable” results The only available analyses of genetic differentiation
for genetic differentiation. Hale et al. (2012) stated that the within Italian white oak populations including Q. pubescens
number of individuals per population should be established s.l. carried out using EST-SSRs markers are those reported
using a “case by case” approach. On the other hand the ge- for the mixed Q. petraea, Q. frainetto, Q. pubescens woods of
netic literature on this issue shows a wide range of opinions the Mount Vairano range in southern Italy (Antonecchia et
and experiences from which it emerges that the number of al. 2015). Comparison between these analyses and our data
individuals sampled can be highly variable. In many cases revealed that the genetic variability of the Apulian popula-
it was considered as acceptable to take a minimum of 10 to tions is significantly lower than that recorded in Mount Vai-
30 individuals. For some Pinus sylvestris L. populations in rano (aHe = 0.72, k = 9.46 Ar = 9.2) as regards aHe and Ar,
the Baltic area, the number of 20-25 individuals was found and slightly higher as regards k.
to be large enough to detect all the alleles (Danusevičius et Also the level of genetic variability among the Q. pube-
al. 2016) whereas other studies have even considered sig- scens s.l. populations of the Apulia region (FST = 0.036) was
nificantly lower numbers (Kitamura et al. 2017, Rinaldi et found to be lower than the FST values found for other oak
al. 2019). In contrast, in Kalinowski (2005) it was shown species in other parts of the world, e.g. Q. rubra L. (FST =
particularly clearly that when FST is less than 0.01 it can be 0.080) (Sullivan et al. 2013), Quercus mongolica Fisch. ex
useful to sample up to 100 individuals. As far as we are con- Ladeb. (FST = 0.077) (Ueno and Tsumura 2008), Q. variabi-
cerned, we believe that these results, although deriving from lis Bl. (FST = 0.063) (Shi et al. 2017).
ACTA BOT. CROAT. 79 (1), 2020 21
DI PIETRO R, DI MARZIO P, ANTONECCHIA G, CONTE AL, FORTINI P
As regards gene flow, the present study showed that the
Apulian Q. pubescens s.l. populations are characterized by a
relatively high values (Nm = 6.664). Although Q. pubescens is
a typical wind-pollinated and outcrossing species such high
values are quite surprising considering the progressive con-
traction suffered by the Apulian oak woods in the last mil-
lennium. The value of gene flow is determined by many fac-
tors, such as the biological means of spreading via pollen and
seeds, the occurrence of physical barriers among populations,
population dimension and so forth; in addition, it can be fa-
cilitated by physical proximity of the populations. It is prob-
able that the short distance separating some of the Apulian
pubescent oak populations and the lack of significant moun-
tain barriers have contributed to keep the average gene flow
value high. It is not possible, at present, to establish whether
the above gene-flow values are stable over time, or are the
result of a contraction due to over-exploitation of forests. In
studies on other oak species (Shi et al. 2017) it was established
that gene-flow values can decrease significantly in a relatively
short time (ten years) following the transition from primary
forest to natural secondary forest. In the Apulia region the
millennial exploitation of the woodlands led to the disappear-
ance of primeval forests as early as Roman times. However,
the secondary forest communities that have since replaced
the primary ones show a high degree of naturalness and a
rather stable floristic composition. It is conceivable therefore
that the gene flow rate will remain stable over time and that
the only parameter capable of negatively acting on the gene-
flow rate is the spatial reduction of the population size.
The apparent relatively low level of intraspecific diversi-
ty observed for the Apulian Q. pubescens populations could
be addressed to their marginal geographical position not
only in the Italian Peninsula but also in the central Med-
iterranean context. In terms of biological conservation of
deciduous oak forests the Apulia region is particularly ex-
posed to environmental risks due to climate change drying
effects which overlap a bioclimatic pattern dominated by the
Thermo-Mediterranean thermotype the latter having in the
Mediterranean maquis the main type of potential vegeta-
tion (Rivas-Martínez et al. 2004, Ladisa et al. 2012). Threats
to pubescent oak woods are further increasing in many lo-
calities that are experiencing greater aridity and water defi-
ciency due to the overexploitation of water reserves result-
ing from the ever increasing consumption for agricultural
purposes and by tourist settlements. In the long term, the
combined effect of these two factors could lead to a change
in the floristic composition of the upper structural layers of
the thermophilous deciduous oak forests with a progressive
replacement of the pubescent oak individuals by evergreen
woody species, such as Quercus ilex L. Phillyrea latifolia L.,
Rhamnus alaternus L., Arbutus unedo L. and Pistacia lentis-
cus L. Conversely, the high gene flow values observed in the
study area seem to move in the opposite direction and might
positively contribute to preventing possible activations of ge-
netic drift. Yet the lack of bottleneck effects in all investigated
populations implies a certain degree of resilience despite the
relatively small areas covered by the single oak populations.
22 ACTA BOT. CROAT. 79 (1), 2020
As regards possible taxonomic implications arising from
this study, the results of STRUCTURE Bayesian clustering
showed that the populations of Apulian pubescent oaks
could not be divided into groups. None of the populations
investigated displayed significant differences in their genetic
composition. However, the highest percentage of the possi-
ble “putative hybrids” that we simply named “off-types”, was
found in Pop13, which also showed the highest percentage of
private alleles. Pop13 is located within a flat sub-humid area
in the western side of the Murgian plateau in the municipal-
ity of Laterza, and lies in spatial contact with a small stand
of Q. robur wood (the only known site of Q. robur currently
recorded for the Apulia region). It is conceivable that in this
area Q. robur was much more abundant in the past and that
both climate change and the millennial work of deforesta-
tion carried out by the local populations, has led to its cur-
rent extreme impoverishment. It is very probable that during
the climatic oscillations that took place during the Quater-
nary, environmental situations favorable to the development
of mixed forests of Q. robur and Q. pubescens s.l. were cre-
ated several times with consequent hybridization or intro-
gression between these two species. In fact, a possible key of
interpretation of genetic structure of the Q. pubescens Apu-
lian populations cannot fail to consider some physiographic
and geographical features of this region such as the lack of
sizeable mountain systems and its deep wedges jutting into
the Adriatic Sea. These features led to the Apulian Peninsula
experiencing the effects of quaternary glaciations only mild-
ly and allowed some parts of it to work as glacial refuges for
the thermophilous oak forests during the Quaternary cold
periods, when most of the Italian Peninsula was covered by
steppic grasslands (Follieri et al. 1988). The occurrence of
restricted areas where the climate forced the various species
of white oaks to coexist for long (cold) periods may have fa-
vored the generation of hybrids. The innumerable past hy-
bridization or introgression events, which led to the current
high morphological variability of Q. pubescens s.l., may also
have played a role in enlarging the gene flow of the Apulian
populations. White oaks commonly live in sympatry, mak-
ing them particularly suitable for gene exchange and pro-
duction of hybrid individuals (Rushton 1993, Williams et al.
2001, Lepais and Gerber 2011). According to Burger (1975),
applying the biological concept of species to the genus Quer-
cus would cause an upheaval of the nomenclature, since the
binomial species would then not correspond to the biologi-
cal one. This makes the systematics of this genus extremely
susceptible to disagreement among botanists and the hope
of arriving at the proposal of a largely shared and possibly
unambiguous taxonomic framework (in particular for the
south-European white oaks) is something not soon to be re-
alized. The problematic classification of pubescent oaks in
the Apulian Peninsula and in the entire Italian Peninsula is
perfectly evidenced by the discrepancy that emerges when
comparing the taxonomy of the genus Quercus as reported in
latest versions of Flora d’Italia (Pignatti et al. 2017) and in the
Checklist of the Italian vascular flora (Bartolucci et al. 2018).
Although both works are worthy of the utmost respect and

consideration, they show enormous differences in both the
numbers and the names of the taxa considered good species
for the same taxonomic group (white oaks). Actually, based
on what emerges from our work, the high taxonomic dif-
ferentiation among the Italian pubescent oaks as reported
in most of the Italian floras and checklists does not seem to
have much foundation (see also Di Pietro et al. 2012) or at
least does not seem to have it with regard to the pubescent
oaks of Apulian Peninsula. In fact, our results established
that it was not possible to identify genetic clusters among
the pubescent oak populations of the Apulian region basing
on 11 highly polymorphic markers as it was not possible to
identify morphological clusters basing in a previous biomet-
ric study on the morphological traits of leaves and fruits on
the same set of specimens (Di Pietro et al. 2016).
Summarizing, the presence of more than one taxon at
species rank belonging to Q. pubescens s.l. (e.g., Q. virgiliana,
Q. dalechampii, Q. amplifolia, etc.) is not confirmed (at least at
present) for the Apulia region. At the same time, the phytoso-
ciological frameworks that are currently supposed to classify
different types of pubescent oak forests (phytosociological
associations), dominated by different pubescent oak species
and including up to five different species of pubescent oaks
in the dominant tree layer, need at the very least to be recon-
sidered. Obviously we do not consider this work to be a so-
lution to all the problems concerning the taxonomy of white
pubescent oaks, not even to some of them of topical relevance
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